Development and Application of Transcriptome-Derived Microsatellites in Actinidia eriantha (Actinidiaceae)

Abstract

Actinidia eriantha Benth. is a diploid perennial woody vine native to China and is recognized as a valuable species for commercial kiwifruit improvement with high levels of ascorbic acid as well as having been used in traditional Chinese medicine. Due to the lack of genomic resources for the species, microsatellite markers for population genetics studies are scarce. In this study, RNASeq was conducted on fruit tissue of A. eriantha, yielding 5,678,129 reads with a total output of 3.41 Gb. De novo assembly yielded 69,783 non-redundant unigenes (41.3 Mb), of which 21,730 were annotated using protein databases. A total of 8,658 EST-SSR loci were identified in 7,495 unigene sequences, for which primer pairs were successfully designed for 3,842 loci (44.4%). Among these, 183 primer pairs were assayed for PCR amplification, yielding 69 with detectable polymorphism in A. eriantha. Additionally, 61 of the 69 polymorphic loci could be successfully amplified in at least one other Actinidia species. Of these, 14 polymorphic loci (mean N_A = 6.07 ± 2.30) were randomly selected for assessing levels of genetic diversity and population structure within A. eriantha. Finally, a neighbor-joining tree and Bayesian clustering analysis showed distinct clustering into two groups (K = 2), agreeing with the geographical distributions of these populations. Overall, our results will facilitate further studies of genetic diversity within A. eriantha and will aid in discriminating outlier loci involved in local adaptation.

Keywords: Actinidia eriantha; EST-SSRs; high-throughput sequencing; population genetic structure; transcriptome.

Figure 1

Outline of the process of unigene annotation and EST-SSR development in Actinidia eriantha.

Figure 2

Distribution of unigene lengths resulting from de novo transcriptome assembly of fruits from Actinidia eriantha.

Figure 3

Gene Ontology classification of unigenes. Unigenes were assigned to three categories: cellular component, molecular function, and biological process.

Figure 4

KEGG pathway assignment using a reciprocal BLAST analysis with a E-value cutoff of 1e–5.

Figure 5

NJ tree and Bayesian clustering analysis results for 14 EST-SSRs of 186 individuals (7 populations) of Actinidia eriantha from South China and East China. (A) Histogram of the STRUCTURE analysis for the model with K = 2 (showing the highest delta K). Each individual is represented by a single vertical line. On the y-axes is the likelihood of assignment to each cluster. (B) Geographic origin of the seven populations and their color-coded grouping according to the STRUCTURE analysis. Population codes are identified in Table 1. (C) The neighbor joining tree of the seven populations with bootstrap values indicated in nodes with support (>50).